Summary of Study ST002792

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001740. The data can be accessed directly via it's Project DOI: 10.21228/M8SQ7X This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002792
Study Title	Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria
Study Summary	All current treatments for malaria are threatened by drug resistance, and new drug candidates that act on novel pathways are urgently needed. Here, we describe MIPS2673, a selective inhibitor of the Plasmodium M1 alanyl metalloaminopeptidase, which displays excellent in vitro antimalarial activity with no significant host cell toxicity. Biochemical assays revealed potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (Pv-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases. Orthogonal chemoproteomic methods based on thermal stability and limited proteolysis reproducibly identified PfA-M1 as the sole target of MIPS2673 in parasites from approximately 2,000 detected proteins. Furthermore, the limited proteolysis approach enabled estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Functional investigation by untargeted metabolomics further demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our proteomics and metabolomics target deconvolution strategies provided unbiased confirmation of the on-target activity of a PfA-M1 inhibitor, and validated selective inhibition of this enzyme as a promising multi-stage and cross-species antimalarial strategy.
Institute	Monash University
Last Name	Siddiqui
First Name	Ghizal
Address	381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Email	ghizal.siddiqui@monash.edu
Phone	99039282
Submit Date	2023-07-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-08-10
Release Version	1

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:	SP002905
Sampleprep Summary:	Pf3D7 cultures underwent double sorbitol synchronization 14 h apart, followed by further incubation for 28-42 h to achieve the desired trophozoite stage (28 hpi) at 6% parasitaemia and 2% hematocrit. Infected RBCs (2x108) were treated with 10x the EC50 of MIPS2673 for 1 h, after which metabolites were extracted. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Sampleprep ID:

SP002905

Sampleprep Summary:

Pf3D7 cultures underwent double sorbitol synchronization 14 h apart, followed by further incubation for 28-42 h to achieve the desired trophozoite stage (28 hpi) at 6% parasitaemia and 2% hematocrit. Infected RBCs (2x108) were treated with 10x the EC50 of MIPS2673 for 1 h, after which metabolites were extracted. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.